PROJECT SUMMARY Certain neurosteroids are effective modulators of g-aminobutyric acid-A receptors (GABAARs), augmenting the actions of GABA at low concentrations and directly activating GABAAR chloride channels at higher concentrations. Present evidence indicates that GABAAR potentiation is a key mechanism in the antianxiety, antidepressant, sedative and anticonvulsant actions of neurosteroids. We have developed novel tools that have helped us to probe the effects of neuroactive steroids, including a series of steroid enantiomers and fluorescent steroids. These agents demonstrate that membrane and cellular accumulation participate in the kinetics and potency of neurosteroids on GABAARs and modulate a specific form of inhibition involving proximal inhibitory inputs near neuronal cell bodies. We have also developed tools for probing the roles of endogenous neurosteroids. Despite these advances, key questions remain with regard to the biological basis and the physiological roles of various steroid pools. In the present proposal, we will address the following aim: To determine the roles of membrane and intracellular pools of neurosteroids on GABAAR modulation, focusing on a form of inhibition resulting from stimulation of GABAergic inputs to the somatic regions of hippocampal pyramidal neurons. These studies will use a combination of physiological and cellular imaging methods to examine steroid effects in cultured rat hippocampal neurons and hippocampal slices, and will involve mechanistic studies based upon our initial observations with novel enantiomeric fluorescent steroid analogues and photoaffinity labels that can be used for cellular physiology and imaging. These studies will provide new information about neurosteroid effects in the CNS and a better understanding of steroid-mediated GABAAR modulation and neuropsychiatric effects.